Rhamnose Mediated Characterization

Introduction

Some well-known promoters like the Lac promoter and the Tet promoter have a repressor that can be inactivated by a chemical, making characterization relatively easy. In this way the promoter can be induced by this chemical. By treatment of different concentrations of this chemical there will be different expression levels of this promoter. With this information you can compare different promoters. The drawback of this is that you need a promoter that is inducible with a chemical. There are also repressors and promoters where that is not possible such as the CIλ promoter and repressor. The CIλ repressor is not inhibited by a chemical so the CIλ promoter is not inducible. To characterize non inducible promoters we developed a new system to characterize promoters based on a two plasmid system with a ramose promoter and GFP, see Figure 1.

characterization design

The final design to characterize promoters includes the following two components:

• A repressor plasmid with a rhamnose promoter and a repressor of choice
• A promoter GFP plasmid with your favoured promoter and GFP

The double repressible promoters used in the kill switch system are taken from the iGEM Registry. Since these promoters pCI/lac and pCI/tet were not characterized we could not estimate if a functional kill switch could be constructed with these promoters. The double repressible promoter pCI/lac from the toggle switch, is characterized. pCI/lac was combined with a gfp reporter gene under Elowitz RBS (BBa_I13504 ) to make the new BioBrick pCI/lac gfp . As a reference pRha and the tetracyclin promoter pTet are also assembled to the gfp gene (BBa_K1493501, BBa_K1493504 ). Since the promoter strength of pTet is known in relative promoter unit (RPU) it can be used as a reference promoter to estimate promoter strength [1]. In this way, we can compare the constitutive promoter strength at one time point and give the characterized promoter a RPU value. The rhamnose promoter was successfully assembled to the CI (BBa_P0451) and lac (BBa_P0412) repressor protein genes under Elowitz RBS creating new BioBricks BBa_K1493510 and BBa_K1493520.

Results

The graphs containing all data points can be found in here. The characterization protocol can be found here.

The measurements in figure 2 indicate an activation of pRha by L-rhamnose. The RFU values of 0% and 0.001% rhamnose are not significant taking into account the high standard deviation for these measurements as can be seen in figure 2B. From a rhamnose concentration of 0.01% to 0.2% a significant increase in fluorescence is measured. Fluorescence from figure 2 can be used to predict the concentration of repressor protein produced. Data points for time 8.15 were chosen for the graph in figure 2B due to the peak at time for 0.2% rhamnose, which is visible in figure 2A. This peak can be explained by the rhamnose depletion, as it is consumed by E. coli, causing the stop of promoter induction. We assume that at this time point the highest concentration of repressor proteins are present in the cells inhibiting the expression of GFP.

The constitutive promoter strength of pCI/lac , is determined using the results of the fluorescence measurements. The RFU values of NEB5α E. coli strains containing pCI/lac GFP and pTet GFP are shown in figure 3. As can be seen in this figure pTet is a stronger promoter than pCI/lac. Using the results shown in figure 3 the RPU value of pCI/Lac is determined using the known value of pTet as stated by Kelly et al. 2009 [1]. Data from time point 8.13 was chosen for calculation. Compared to the pTet RPU value 1.5, pCI/lac has a RPU of 1.0.

Figure 4 shows that a higher concentration of L-rhamnose gives a lower RFU value. This means that the GFP expression is repressed by the repressor protein produced under the rhamnose promoter. As shown in figure 15 a low concentration of rhamnose (0.001%) does not have any substantial effect on the expression rate of the protein. This is also visible in figure 4 in which for both CIλ (A) and LacI (B) 0.001% rhamnose doesn’t show a lower fluorescence compared to 0% rhamnose. We expected the same RFU values for cultures grown in medium with 0.2% glucose and 0% rhamnose, since glucose functions as a repressor for pRha. Though, cultures grown in medium with glucose show lower RFU values in both graphs in figure 4. This can be explained by the higher growth rate of the cells in glucose containing medium, since the RFU value is OD dependant (notebook). In figure 15 0.01% rhamnose shows a higher RFU compared to the culture grown on 0% rhamnose, which indicates that pRha is active at that concentration at that time point. However, at the concentration of 0.01% rhamnose both CIλ (A) and LacI (B) don’t show any substantial repression. We can conclude that the concentration repressors produced at the promoter strength of pRha in 0.01% rhamnose is not high enough to perform repression of the promoter pCI/lac. Though, cultures grown in 0.05% and 0.2% rhamnose show a lower RFU value for both constructs as the RFU in figure 15 increases in higher rhamnose concentrations. We can conclude that repression of pCI/lac is stronger in higher concentrations rhamnose, since higher concentrations rhamnose lead to a higher concentration of repressor proteins. We expected the same RFU values for cultures grown in medium with 0.2% glucose.

References

1. Kelly, J.R., et al., Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng, 2009. 3: p. 4.